This invention relates to a unique class of fibroblast growth factor receptors, nucleic acids encoding same and expression of the growth factor receptors in recombinant systems. This invention also relates to the use of the expressed receptors or fragments thereof in screens for candidate drugs which act as receptor antagonists.
The fibroblast growth factor (FGF) family consists of seven related heparin-binding proteins, which include acidic FGF (aFGF), basic FGF (bFGF). int-2, hst/kFGF, FGF-5, FGF-6 and KGF. The members of the FGF family share approximately 30-55% amino acid sequence identity, similar gene structure, and are capable of transforming cultured cells when overexpressed in transfected cells. The prototypic FGFs, aFGF and bFGF were the first to be purified, sequenced and cloned. They are mitogens, in vitro, for a variety of cells of mesenchymal and neuroectodermal origin. In vivo, FGFs can induce the formation of mesoderm in developing Xenopus embryos and possess potent angiogenic activity (reviewed by Burgess and Maciag, Ann. Rev. Biochem. 58: 575-606 (1989)).
The response of cells to FGFs is mediated by binding and activation of specific cell surface receptors possessing intrinsic tyrosine kinase activity. Receptors are proteins, often glycosylated, which serve as integral components of cellular membranes. Typically, receptors possess an extracellular domain located at the cell surface capable of specific interaction with substances known as ligands. The fibroblast growth factors are examples of ligands. As a consequence of the binding of the ligand to the extracellular domain, a second region of the receptor located on the intracellular surface of the membrane (i.e. the cytoplasmic domain), is activated to permit reaction with other intracellular molecules. The cytoplasmic domain comprises a catalytic region, that is a region possessing enzymatic activity. With particular reference to the fibroblast growth factor receptor described herein the catalytic domain is a protein tyrosine kinase. The substrate of this kinase can be the receptor itself (i.e. autophosphorylation) or other intracellular proteins such as phospholipase C-xcex3. The kinase domain of certain receptors can be interupted by insertion of up to 100 mostly hydrophilic amino acid residues. This insert may act to modulate receptor interaction with certain cellular substrates and effector proteins. The cytoplasmic domain terminates with a COOH-terminal tail region. This sequence is the most divergent among all known RTKs. Several autophosphorylation sites have been mapped to this region and the region may act by exerting a negative control on kinase signalling function. The catalytic region is separate from membrane proper by a juxtamembrane domain. Present evidence supports the notion that this region is involved in modulation of receptor function by heterologous stimuli, a process known as receptor transmodulation. Linking the extracellular and cytoplasmic domains is a region spanning the membrane proper known as the transmembrane domain. The ligand interaction is thought to activate the cytoplasmic domain by inducing a conformational change such as by aggregation or other mechanisms. Accordingly, a receptor acts as a molecular transducer, translating an extracellular event (ligand binding) into an intracellular response (cytoplasmic enzymatic activity).
As mentioned above, the substance which is bound by the receptor is known as the ligand; a term which is definitionally meaningful only by reference to its counterpart receptor. Accordingly, the term xe2x80x9cligandxe2x80x9d does not imply any particular molecule size, structural or compositional feature other than that the substance is capable of binding or otherwise interacting with the receptor in such a manner that the receptor conveys information about the presence of the ligand to an intracellular target molecule. Such a functional definition necessarily excludes substances which may bind to an extracellular domain but fail to affect receptor activation. Stated directly not all substances capable of binding receptors are ligands, but all ligands are capable of binding a receptor.
As mentioned above, receptors have been identified that have assayable biological activity dependent on ligand interaction. Generally, the activity is enzymatic and is localized in the cytoplasmic domain. One group of receptors relevant to this invention possess intrinsic protein tyrosine kinase (PTK) activity.
FIG. 1 presents a schematic representation of several known growth factor receptors that bear PTK activity. Growth factor receptors with PTK activity, or receptor tyrosine kinases (RTKs), have a similar molecular topology. All possess a large glycosylated, extracellular ligand binding domain, a single hydrophobic transmembrane region, and a cytoplasmic domain which contains a PTK catalytic domain. Because of their configuration, RTKs can be envisioned as membrane-associated allosteric enzymes. Unlike water-soluble allosteric enzymes, RTK topology dictates that the ligand binding domain and PTK activity are separated by the plasma membrane. Therefore, receptor activation due to extracellular ligand binding must be translated across the membrane barrier into activation of intracellular domain functions.
On the basis of sequence similarity and distinct structural characteristics, it is possible to classify these receptors into subclasses (FIG. 1). Structural features characteristic of the four subclasses include two cysteine-rich repeat sequences in the extracellular domain of monomeric subclass I receptors, disulfide-linked heterotetraineric xcex12xcex22 structures with similar cysteine-rich sequences in subclass II RTKs, and five or three immunoglobulin-like repeats in the extracellular domains of subclass III and IV TRKs, respectively. The tyrosine kinase domain of the latter two subclasses is interrupted by hydrophilic insertion sequences of varying length. The availability of RTK cDNA clones has made it possible to initiate detailed structure/function analyses of the mechanisms of action of RTK family members. (Reviewed by Ullrich and Schlessinger, Cell 61: 203-221 (1990))
This invention is predicated on the discovery of a partial human cDNA clone of a fms-like gene (flg) which encoded a protein tyrosine kinase whose kinase domain was interrupted at a position similar to the kinase inserts of the CSF-1 and PDGF receptor tyrosine kinases (Ruta et al., Oncogene, 3: 9-15 (1988)). Subsequently, full length cDNAs for chicken flg were isolated by cloning with flg-specific oligonucleotide probes (Lee et al., Science, 245: 57-60 (1989)) and with antiphosphotyrosine antibodies (Pasquale and Singer, Proc. Nat""l. Acad. Sci. USA, 86: 5449-5453 (1989)). Flg is an FGF receptor based on four criteria: 1) the receptor purified from a chicken embryo extract by affinity chromatography on immobilized bFGF is the chicken flg gene product, 2) flg anti-peptide antiserum immunoprecipitates [125I]aFGF crosslinked to proteins of 130 and 150 kDa in A204 rhabdomyosarcoma cells, 3) flg-anti-peptide antiserum immunoprecipitates proteins of similar size which are specifically phosphorylated on tyrosine upon treatment of living cells with aFGF or bFGF, and 4) proteins of 130 and 150 kDa immunoprecipitated with flg anti-peptide antiserum from aFGF-treated cell lysates undergo tyrosine phosphorylation in vitro.
A putative second FGF receptor is the mouse bacterially expressed kinase (bek) gene product, a partial clone of which was obtained by screening a mouse liver cDNA expression library with anti-phosphotyrosine antibodies (Kornbluth et al., Mol. Cell. Biol., 8: 5541-5544 (1988)). The deduced amino acid sequences of the partial bek clone and the corresponding region of flg are 85% identical. However, it was unclear whether bek represents the mouse homologue of the human flg gene or another closely related gene. This invention provides full length cDNA clones for both human bek and flg, their complete deduced amino acid sequences, and demonstrates in transfected cells that both bek and flg bind aFGF, bFGF and k-FGF specifically and with high affinity.
Receptor function has increasingly become the focus of attempts to rationally design drugs. Molecules that affect receptor-ligand binding, receptor activation and/or receptor intracellular target interaction are all potential candidates for therapeutics. Large scale screening is limited by conventional methods in which candidate drugs are tested on isolated cells or tissue explants. Tissue samples or isolated cells containing the target receptors are costly to obtain, present in limited quantity and difficult to maintain in a functionally viable state. Additionally, it is often difficult to reliably and reproducibly administer the candidate drug to tissue samples. Screening assays using primary explants in tissue culture are undertaken in larger scale than is possible with tissue samples. However, it is more difficult to assay physiological effect and the assays are subject to interference from many sources, e.g. culture media or cultivation conditions. Finally, assays using receptors isolated from natural materials have the disadvantage that the receptor is subject to natural variability and suitable natural sources may not always be available. It is an object herein to provide receptor molecules in a form amendable for large scale screening protocols.
This invention provides a receptor protein or fragments thereof, in isolated form, comprising a cytoplasmic domain with protein kinase activity, a kinase insert, a transmembrane domain and an extracellular domain having three immunoglobulin-like repeats and biologically active equivalents thereof.
Another aspect of this invention is an isolated DNA sequence encoding a receptor protein or fragments thereof, said protein having a cytoplasmic domain with protein kinase activity, a kinase insert, a single transmembrane domain and an extracellular domain having three immunoglobulin-like repeats and biologically equivalents thereof.
Another aspect of this invention is a vector comprising a cDNA encoding a receptor protein, said protein having a cytoplasmic domain with protein kinase activity, a kinase insert, a single transmembrane domain and an extracellular domain having three immunoglobulin-like repeats.
Another aspect of this invention is a host cell transformed with the above-described vector.
Another aspect of this invention is a therapeutic composition comprising the extracellular domain or fragment thereof of a human receptor protein comprising a cytoplasmic domain having protein kinase activity, a kinase insert, a single transmembrane domain and an extracellular domain having three immunoglobulin-like repeats in an amount effective to inhibit undesirable heparin-binding growth factor mediated cellular responses and a pharmaceutical acceptable carrier.
Another aspect of this invention is a therapeutuc composition comprising the extracellular domain or fragment thereof of a human receptor protein comprising a cytoplasmic domain having protein kinase activity, a kinase insert, a single transmembrane domain and an extracellular domain having three immunoglobulin-like repeats in an amount effective to inhibit the binding of an opportunistic pathogen to human cells bearing said receptor protein and a pharmaceutical acceptable carrier.
Another aspect of this invention is a method for the treatment of a patient with a disease characterized by an undesirable heparin-binding growth factor mediated cellular response comprising administering to such patient an effective amount of the composition.
Another aspect of this invention is a method for the treatment of a patient suffering from an opportunistic pathogen infection said pathogen being capable of binding to a heparin-binding growth factor receptor comprising administering to such patient an effective amount of the composition.
Another aspect of the invention is a method for the screening of drugs that inhibit fibroblast growth factor binding to cellular receptors comprising the steps of:
(a) incubating the drug with host cells capable of overexpressing fibroblast growth factor receptors in the presence of a fibroblast growth factor and
(b) measuring the ability of the drug to inhibit fibroblast growth factor binding.
A final aspect of the invention is a method for screening of drugs that inhibit the binding of opportunistic pathogens to fibroblast growth factor receptors comprising the steps of:
(a) incubating the drug with host cells capable of overexpressing fibroblast growth factor receptors in the presence of said pathogen and
(b) measuring the ability of the drug to inhibit pathogen binding.